Project Summary The goal of this proposal is to develop a widely adoptable, high-throughput, functional connectomics platform to semi-automatically reconstruct and analyze the synaptic connectivity of functionally characterized neuronal microcircuits. We will develop this pipeline in the context of understanding neural microcircuits that control walking, using the Drosophila ventral nerve cord (VNC) as a model system. The VNC constitutes over a third of the fly central nervous system, and functions like the spinal cord to control movement and sensation of the limbs. It has a tractable number of neurons (~60,000), many of which are genetically identified and anatomically stereotyped, making it an excellent brain region for developing a comprehensive approach to characterize neuronal circuits. Our approach will integrate several technologies from our labs, including in vivo two-photon calcium imaging in walking flies, high-throughput transmission electron microscopy (EM), automated, deep- learning based connectomic reconstruction, and cell morphology-based analytics. Using this pipeline we will generate the first dense functional connectomes of the VNC, including the sensorimotor leg circuitry from multiple male and female flies. We will use these data to test the hypothesis that there exist specific patterns of synaptic connectivity between functional subtypes of leg proprioceptors and motor neurons. By creating the first connectomes of a microcircuit that orchestrates walking behavior, we anticipate this project will provide new and fundamental insight into the circuit basis of sensorimotor transformation and motor control. It will also create an important resource for the Drosophila and neuroscience communities, through publicly available tools and datasets that integrate connectivity, morphology, and cellular physiology data. Moreover, the technical approaches we develop for high-throughput functional connectomics can be readily applied to other brain regions and across species.